One of the most sensitive methods for detecting small quantities of materials, is the immunoassay method, which is based on the combination of the material to be detected, usually called an antigen, and an antibody, raised in an animal, to that antigen. Either the antigen or the antibody is labelled in some fashion to allow for detection. From being a research tool, the method is now used routinely for the screening of large numbers of samples, especially human plasma, for pathogens and foreign substances, such as drugs.
The combination of antigen and antibody, which usually gives a complex of lower solubility than either of the two starting materials, in a test system, is detected by a variety of methods. Initially detection was achieved by radioactively labelling one of the reactants and determining the amount of radiation, either in the separated antigen-antibody complex or in the solution after removal of the complex. More recently detection has been achieved by labelling one of the reactants with an enzyme, which can cause a colour change in the test system, when a substrate is added. A well known example of such an enzyme is horse radish peroxidase, and the test system is known as the enzyme-linked immunosorbent assay (ELISA).
This principle of using an antigen-antibody complex is employed in a number of assays for the detection of pathogenic organisms, or their products, which have invaded a mammalian organism. The method can also be used for the detection of antibodies which the organism has raised to the pathogen. This latter assay is used to determine whether the mammalian organism has been previously exposed to the disease. Examples of these two assays are the detection of the Hepatitis B surface antigen (see, for example, U.S. Pat. Nos. 3,867,517 and 4,197,361) and antibody to this antigen (U.S. Pat. No. 4,230,683).
A number of different types of immunoassay, using the antigen-antibody complex principle, have been developed. One of the most useful immunoassays is the so-called `sandwich-type` assay, in which one reactant is bound to a solid-phase, for example, a plastic tube, bead or microplate well. This complex is exposed to the second reactant and this mixture, after washing, again is exposed to the first reactant, which has been labelled in some fashion. From this sequence of operations the quantity of the second reactant in the sample can be determined by determining the quantity of labelled material. The second reactant is sandwiched between two layers of the first. Habermann [Z.Clin.Chem.Clin.Biochem.,8,51,(1970)] used the method for the detection of tetanus toxin present in test samples. The sandwich immunoassay also has been successfully used for the detection of the five different types of antibody present in human and animal sera.
In all the above techniques, the sandwich is built up sequentially by contacting the individual components with the underlying solid-phase for a period of time and then any unbound material is washed off. At no time has a mixture of antibody and antigen been used, since it is generally considered that the formation of the antibody-antigen complex diminishes the immunoreactivity of both components.